Nanophotonics technology heralds advances for applications in nanomedicine, where the generation of induced Pluripotent Stem cells (iPS) represents a promising key enabling technology. Current preferred technology to achieve the latter consists of viral based solutions introducing the so called pluripotency genes. However, their application is well-known to present increased mutagenesis risks. Liposomes on the other hand are considered as a promising carrier to drug and gene delivery, but currently don’t achieve viral vector reprogramming efficiencies. Here, we present the application of Two-Color Fluorescent Cross-Correlation Spectroscopy (2c-FCCS) as a remarkable tool to analyze and potentially optimize the load-efficiency of DNA-liposome complexes for the reprogramming of human fibroblasts into iPS (Figure 1).
Knowing that the composition of liposomes can directly affect cell toxicity and uptake, we initially tested different lipid combination of DOPC, DOTAP and C18-ceramide (Cer). These liposomes not only exhibit better cell uptake in comparison to the commercial transfection reagent Lipofectamine but also exhibited low toxicity (Figure 2). Aided by 2c-FCCS we can propose Cer-based lipoplexes as good choice for iPS generation since they obtain higher load-efficiency for some of the plasmids (Figure 3). Based on our work we suggest the use of 2c-FCCS as a direct method to monitor and potentially optimize the formation of new lipoplex formulations.
Figure 1. Overview of lipoplex formation optimizaiton through 2c-FCCS: only if both biomolecules co-diffuse there will exist a correlation between them (right). A low or zero cross-correlation amplitude means that liposomes and plasmids diffuse separately through the confocal volume (left).
Figure 2. Cell uptake and death analysis of human fibroblast treated with DiI-liposomes (red): a – e) cell uptake of DOPC/Cer (a), DOTAP/Cer (b), DOPC/DOTAP/Cer (c), Lipofectamine (d) and DOTAP (e). Nucleus labeled with DAPI (blue). (f) Cell death quantification through PrestoBlue assay using H2O2 as positive control. Graph bars represents mean ± SEM, where *p < 0.0001.
Figure 3. Two-Color Fluorescent Cross-Correlation Spectroscopy analysis: a–d) Auto- and cross-correlation FCCS curves using DOPC/Cer for lipoplex formation assessment; f) Graph representation of the loading-efficiency of the different combinations of lipoplex using pluripotent genes.
Enhanced spectroscopy and sensing , Optical sensing from solid state to bio-medicine
